REVISED ABSTRACT: The overall goal of this Program Project Grant is to elucidate the mechanisms involved in the metal homeostasis by human cells. Copper and iron are essential co-factors in a wide variety of biological processes and while low regulated levels of these metals are essential, elevated levels are toxic. The physiological handling of copper can be viewed in several steps. These are, uptake into the cell, delivery to specific copper-dependent proteins and removal from the cell. The molecular mechanisms of copper distribution will be studied at the molecular, cellular and tissue level in an interdisciplinary approach. Uptake, in humans, is mediated by hCtrl, and one of the PPG projects will carry out a detailed characterization of this recently discovered protein. The second PPG project will perform a structural and mechanistic analysis of specific delivery to copper dependent proteins by copper chaperones. The removal of copper is accomplished by copper-activated ATPases that are regulated by copper at several levels. In Project 3, a recently discovered copper-dependent phosphorylation of one of these ATPases will be used as a model to understand the basis of copper signaling and regulation. The PPG core including confocal microscopy, mass spectrometer, and other instrumentation, facilitates this multi-level strategy. Disruptions in metal homeostasis are being increasingly invoked as the basis for many neurodegenerative and other life-threatening diseases. In order that new and more effective therapies can be discovered it is necessary to have a clearer understanding of the regulation of metal ions in normal states. The goal of this program is to initiate a systematic investigation of these processes and provide such information to the biomedical community.